Abstract Francisella tularensis, the aetiological agent of tularemia, is one of the most infectious bacterial pathogens currently known and a NIAID category A priority pathogen because of its potential for use as a biological weapon. In F. tularensis, the SspA protein family members MglA and SspA play a critical role in regulating the expression of virulence genes that are essential for intramacrophage growth and survival. MglA and SspA form a complex that associates with RNA polymerase (RNAP) to positively control the expression of virulence genes by a mechanism that is not fully understood. We have found that in the live vaccine strain of F. tularensis, the MglA-SspA complex works in concert with the small molecule ppGpp, and a putative DNA-binding protein we have named PigR, to control the expression of a common set of genes. PigR interacts directly with the MglA- SspA complex and ppGpp promotes this interaction. However, it is not known precisely how ppGpp exerts its regulatory effects. We have also uncovered a small sequence motif that is present upstream of the -35 element of regulated promoters that is necessary and sufficient for PigR (and thus presumably MglA, SspA, and ppGpp) to exert its regulatory effects. We refer to this sequence motif operationally as the PigR response element (PRE), but we do not know how this small sequence motif renders a promoter subject to control by these key virulence regulators. In Aim 1 of this proposal we will test whether ppGpp exerts its effects by binding directly to the MglA-SspA complex, determine the sequence requirements of the PRE, and test whether the PRE serves as a contact site for PigR. RNAP in F. tularensis is highly unusual in that it contains two distinct a-subunits and we have shown that the C-terminal domain of one of the a-subunits (the a2-CTD) specifically influences the expression of MglA-controlled genes. In Aim 2 we will determine how the a2-CTD exerts its effect on the expression of MglA-controlled genes. In Aim 3 we will use next-generation sequencing approaches to beter define the MglA regulon and the contribution of its members to intramacrophage growth. The proposed work is expected to reveal how an important intracellular pathogen regulates the expression of genes required for survival in the host, and has implications for how ppGpp and SspA family members regulate gene expression in other pathogenic bacteria. The proposed studies may also help uncover why the transcription machinery in F. tularensis has an unprecedented a-subunit composition. Finally, the proposed work will provide a more complete picture of the contribution of MglA to intramacrophage growth and will also provide an important resource to the Francisella field by defining all of the bacterial requirements for intramacrophage growth.